Comparative Studies on the Structure and Biological Activities of Two New Polysaccharides from Tricholoma sinoportentosum (TS-P) and Termitomyces albuminosus (TA-P)

Polysaccharides are important active ingredients of living organisms. In this study, two new polysaccharides, Tricholoma sinoportentosum polysaccharide (TS-P) and Termitomyces albuminosus (TA-P), were extracted and purified using anion exchange column chromatography. The structure of each polysaccharide was identified by HPGPC, FT-IR, HPLC, GC-MS and NMR, and the biological activities were also investigated. The results of the structure identification showed that TS-P was composed of arabinose, mannose, glucose and galactose at a ratio of 1:1:3:2 and its main chain was composed of (1→4)-Arap residues, (1→4,6)-D-Manp residues and two (1→6)-Galp residues. The TA-P was composed of arabinose, glucose and galactose at a ratio of 2:4:8. Its main chain was composed of two (1→4)-β-L-Arap residues, one (1→4)-Glcp residues, three (1→2,6)-Galp residues and five (1→6)-Galp residues. The immunoassay showed that TS-P and TA-P could significantly promote the proliferation of T cells, B cells and RAW264.7 cells. The cell cycle results showed that for B cells and macrophages, TS-P and TA-P mainly affected the G0/G1 phases of the cell cycle; for T cells, TS-P affected G2/M phase, while TA-P mainly affected the G0/G1 phases. TS-P could significantly promote B cells to secrete IgA, IgG and IgD (p < 0.01), while TA-P could significantly promote the secretion of IgA and IgG (p < 0.01). The chemical structure and biological activity of TS-P and TA-P were first studied and compared to lay a theoretical foundation for the application of fungal polysaccharide.


Introduction
Currently, chemotherapy is still the main treatment for malignant tumors. Medicine resistance and toxic side effects generated during long-term chemotherapy are the main reasons for chemotherapy failure [1] Therefore, searching for natural products with low toxicity, high efficiency and anti-tumor activity has become a research hotspot. Fungi contain a variety of chemical components, such as polysaccharides, polypeptides, polyphenols, alkaloids, carotenoids, minerals, vitamins, terpenoids, etc. [2][3][4][5], and fungi polysaccharides have become the subject of attention because of their good biological activity, especially their immunomodulatory and anti-tumor effects. As early as the 1960s, Chihara et al. reported on the anti-tumor properties of lentinan [6][7][8]. More and more polysaccharides have been found to have biological activities such as anti-tumor, regulating immunity, anti-inflammatory and anti-diabetes [9,10]. Further study on the structure and biological activity of polysaccharides can greatly promote the research and development of edible and medicinal polysaccharides.

FT-IR Analysis of TS-P and TA-P
The Fourier transform infrared spectrum of TS-P is shown in Figure 1E. A broad and strong absorption peak appeared at 3425 cm −1 and was designated as the stretching vibration peak of O-H; the absorption peaks at 2923 cm −1 and 1639 cm −1 were designated as the stretching vibration peaks of -CH 2 and C=O, respectively; the absorption peak at 1402 cm −1 was designated as the bending vibration peak of the C-H plane; the stretching vibration peaks of C-O appeared at 1079 cm −1 and 1047.174 cm −1 ; the absorption peak at 673 cm −1 was specified as the bending vibration of -CH.
The results of the TA-P infrared spectrum are shown in Figure 1F. The wide and strong absorption peak at 3428 cm −1 was designated as the O-H stretching vibration peak; the peaks at 2925 cm −1 , 1639 cm −1 and 1400 cm −1 were designated as the -CH 2 stretching vibration peak, C=O stretching vibration peak and C-H bending vibration peak, respectively. The C-O stretching vibration peaks were at 1137 cm −1 , 1076 cm −1 and 1043 cm −1 . The signal at 674 cm −1 was the bending vibration peak of =C-H. In addition, there was no absorption peak near 1730 cm −1 , indicating that TA-P does not contain uronic acid. The absorption peaks appearing at 1200-1000 cm −1 indicated that the monosaccharide rings both in TS-P and TA-P were pyran type.

Monosaccharide Composition Analysis of TS-P and TA-P
The HPLC results showed that the retention times of Rha, Xyl, Ara, Fru, Man, Glc and Gal were 4.51, 5.29, 5.83, 6.33, 6.58, 7.41 and 7.70 min, respectively. The HPLC results of TS-P after hydrolysis showed that the polysaccharide had four retention time peaks. Referring to the retention time of monosaccharide standard, it indicated that the peaks at the retention time of 5.29, 6.54, 7.37 and 7.56 min were the peaks of arabinose (Ara), mannose (Man), glucose (Glc) and galactose (Gal), respectively, and their peak area ratio was 1:1:3:2 ( Figure 1G).
The results showed that the retention times of Rha, Xyl, Ara, Fru, Man, Glc and Gal were 4.21, 4.82, 5.29, 5.67, 5.83, 6.43 and 7.43 min, respectively. The HPLC results of TA-P after hydrolysis showed that the polysaccharide had three retention time peaks. The peaks at retention times of 5.20, 6.42 and 7.39 min were the peaks of arabinose (Ara), glucose (Glc) and galactose (Gal), respectively, and their peak area ratio was 1:2:4 ( Figure 1H).

1 H-1 H COSY Analysis of TS-P and TA-P
The 1 H-1 H COSY spectrum can reflect the coupling relationship between adjacent hydrogen nuclei [28]. It solves the problem of partial proton signal overlap in polysaccharide and can obtain each proton signal of monosaccharide in polysaccharide. The 1 H-1 H COSY data of TS-P showed that signals A (δ 5.04/3. (1→4)-Arap (E) groups, respectively. The signals of H2-H6 of the A group were δ 3.85, δ 3.97, δ 3.69, δ 3.75 and δ 3.53, respectively, based on the coupling signal of adjacent 1 H atoms. The 1 H signals of B-E groups were also deduced ( Figure 3E, Table 3 Table 4).

HMBC Analysis of TS-P and TA-P
HMBC data reflect the coupling relationship between 1 H and remote 13 C, which can provide the arrangement order and structural information of the molecular skeleton between monosaccharide residues [30]. In the HMBC data of TS-P ( Figure 2I Table 3).
The TA-P was composed of arabinose, glucose and galactose at a ratio of 2:4:8. Its main chain was composed of two (1→4)-β-L-Arap residues, one (1→4)-Galp residue, three (1→2,6)-Galp residues and five (1→6)-Galp residues. There were three branched chains, which were →1)-Glcp residues connected to (1→2,6)-Galp residues as the terminal group ( Figure 4F).  [16]. However, currently, the primary structures of polysaccharide from Tricholoma sinoportentosum and Termitomyces albuminosus have not been identified. The molecular weight size is a necessary condition for polysaccharides to possess biological activity. The biological activity of polysaccharides usually requires their molecular weight to be within a certain range. The molecular weight of polysaccharides is too low to form advanced polymeric structures that generate activity; however, the larger the molecular weight and volume of polysaccharides, the less conducive they are to crossing multiple cell membrane barriers and entering the organism. Most of the polysaccharides with outstanding biological activity are connected by (1→3) or (1→4) glycosidic bonds. This configuration is conducive to the formation of a three strand spiral configuration, showing high biological activity. If the skeleton structure is mainly connected by (1→6) bonds or other bonds, the biological activity is low [31]. The degree of branch (DB), also known as degree of substitution (DS), also affects the activity of polysaccharides. Only when polysaccharides reach a certain degree of substitution can they have biological activity. Each polysaccharide has an optimal DB, enabling its biological activity to reach an ideal state [32,33]. In our study, the structure identification showed that TS-P (Mw 22,900 Da) was composed of arabinose, mannose, glucose and galactose at a ratio of 1:1:3:2 and its main chain was composed of (1→4)-Arap residues, (1→4,6)-D-Manp residues and two (1→6)-Galp residues. The TA-P (Mw 26,500 Da) was composed of arabinose, glucose and galactose at a ratio of 2:4:8. Its main chain was composed of two (1→4)-β-L-Arap residues, one (1→4)-Glcp residues, three (1→2,6)-Galp residues and five (1→6)-Galp residues. These two polysaccharides, TS-P and TA-P, are identified as new structures after SCIFinder query. Both polysaccharides conformed to the structural characteristics of molecular weight, main chain structure and branching degree, which could have potential biological activities.

Effect of TS-P and TA-P on B Cell Activity In Vitro
B cells are pluripotent stem cells derived from bone marrow and play a huge role in humoral immunity. After recognizing and binding to external antigens through the surface receptors in B cells, extrinsic antigens activate the B cell signal pathway [34]. After a series of transformations, B cells differentiate into plasma cells and memory B cells. The plasma cells produce antibodies to remove pathogens to achieve an immune effect. The analysis of results indicated that the B cell proliferation effect was better than the LPS group (28.30%) when TS-P final concentrations were 1.25 and 2.5 µg/mL and the maximum proliferation rate was 40.47% (TS-P 2.5 µg/mL). The B cell proliferation effect was the best (29.60%) when the final concentration of TA-P was 5 µg/mL ( Figure 5A,B). The observation results of cell morphology indicated that the number of B cells in the blank group is small, in bright spherical shape and in good condition. There was a certain dose relationship between the cell mass and TS-P or TA-P concentration, which was larger than that of the blank group at a final concentration of 1.25-10 µg/mL. The cells in the positive group also had no necrosis, grew well and were larger than those in the blank group ( Figure 5C,D).
Cell cycle refers to the process that a continuously dividing cell goes through from the completion of one division to the completion of the next [35]. A cell cycle consists of G0/G1, S and G2/M phases. Based on the results of the effect of TS-P and TA-P on B cell proliferation, the optimal concentration 2.5 µg/mL of TS-P and 5 µg/mL of TA-P were selected for further study of the effect of TS-P and TA-P on the B cell cycle. The percentage of B cells in the G0/G1 phase decreased, while the percentage of B cells in S phase and G2/M phase increased under the stimulation of 2.5 µg/mL TS-P and 5 µg/mL TA-P. The number of B cells in the G0/G1 phase in the TS-P group significantly decreased from 38.90% to 35.40% compared with the blank group (p < 0.01). In addition, the G0/G1 phase and S phase of the positive group were significantly different from the blank group (p < 0.01). The percentage of G0/G1 phase cells decreased by 4.00%, while the percentage of Cytokines are hormone active protein molecules produced by immune cells after being stimulated by an antigen or mitogen [36,37]. The main function of B lymphocytes is to secrete immunoglobulin, which is the final stage of differentiation sequence occurring in different microenvironments in vivo. When the final concentration of TS-P is 2.5 µg/mL, it can significantly promote B cells to secrete IgA, IgG and IgD (p < 0.01), with secretion amounts of 597.00 µg/mL, 22.60 mg/mL and 484.00 µg/mL, respectively. It can significantly promote B cells to secrete IgM (p < 0.05), and the secretion amount was 2219.00 µg/mL. When the final concentration of TA-P is 5 µg/mL, it can significantly (p < 0.01) promote B cells to secrete IgA and IgG with secretion amounts of 603.00 µg/mL and 22.60 mg/mL, respectively. Compared with the blank group, the positive group can significantly promote B cells to secrete IgA, IgG, IgE, IgD and IgM (p < 0.01). It should be noted that the amount of IgD secreted by B cells stimulated by TS-P and the amount of IgA secreted by B cells stimulated by TA-P were higher than those in the positive group ( Figure 5I-M).

Effect of TS-P and TA-P on T Cells Activity In Vitro
T cells are differentiated from lymphoid stem cells in the thymus. They are the most numerous and complex type of cells in lymphocytes and participate in the cellular immunity of the body. Mature dendritic cells can recognize, process and present antigens to T cells, thus activating adaptive immune response [38]. The results showed that TS-P and TA-P could significantly promote the proliferation of T cells (p < 0.01) at the final concentrations of 1.25, 2.5, 5, 10 µg/mL compared with the blank group ( Figure 6A,B). At the same time, the effect of the positive group on stimulating the proliferation of T cells also showed a significant difference (p < 0.01). The effect was better than that of the positive group (35.40%) when the final concentration of TS-P was 2.5 µg/mL or 5 µg/mL, and the proliferation effect of TS-P was the best at 2.5 µg/mL, the rate of which reached 59.30%. When the final concentration of TA-P was lower than 2.5 µg/mL, the proliferation rate was in direct proportion to it, and vice versa; when the final concentration of TA-P was 2.5 µg/mL, the maximum proliferation rate was 43.90%, which was higher than that in the positive group (29.20%). The observation results of cell morphology showed that the number of T cells stimulated by TS-P and TA-P increased and the cell clusters became larger compared with the blank group. To sum up, TS-P and TA-P can promote the proliferation of T cells in a certain concentration range, different concentrations of polysaccharides had different effects on the cell mass volume and cell number, both polysaccharides had a better proliferation effect on T cells at the final concentration of 2.5 µg/mL, and TS-P was superior to TA-P in stimulating T cell proliferation ( Figure 6C,D).
Based on the results of the effect of TS-P and TA-P on T cells proliferation, the optimal concentration of 2.5 µg/mL of TS-P and TA-P was selected for further study on the effect of TS-P and TA-P on the T cell cycle ( Figure 6E-H). The percentage of T cells in the S phase increased and the percentage of T cells in the G0/G1 phase decreased under 2.5 µg/mL TS-P compared with the blank group with no significant difference. At the same time, the percentage of T cells in the G0/G1 phase in the positive group decreased, and there were also no significant differences, but the percentage of T cells in the S phase increased by 9.30%, showing a very significant difference (p < 0.01); further, the percentage of T cells in the G2/M phase increased significantly (p < 0.05) from 12.0% to 13.1% in the TS-P group compared with the blank group. The percentage of T cells in the G0/G1 phase decreased significantly (p < 0.01) by 4.00% and 6.30%, respectively, under 2.5 µg/mL TA-P and 5 µg/mL LPS compared with the blank group, and the reduction in the positive group was greater than that in the TA-P group. The percentage of T cells in the S phase and G2/M phase of the TA-P and positive group increased, while the S phase and G2/M phase of the TA-P group showed a significant difference (p < 0.05) and the G2/M phase of the positive group showed an extremely significant difference (p < 0.01), increasing from 27.30% to 31.80%, compared with the blank group. These results suggested that the T cell cycle changed after being stimulated by TS-P and TA-P. The difference was that TS-P affected the cell cycle by affecting the G2/M phase-that is, by improving the ability of T cells to divide, while TA-P was mainly by affecting the G0/G1 phase.  The study on the effect of cytokines secreted by T cells is the verification of the functional changes of T cells after proliferation ( Figure 6I). The secretory volumes of TNF-α were 33.70 pg/mL and 50.60 pg/mL when T cells were treated with TS-P (2.5 µg/mL) and TA-P (2.5 µg/mL), respectively, which both showed a very significant difference (p < 0.01). The positive group can significantly promote the secretion of TNF-α by T cells (p < 0.05) with the amount of secretion being 35.00 pg/mL, and it was not difficult to find that the TA-P group was better than the positive group. The above results indicated that TS-P and TA-P could not only promote the proliferation of T cells but also promote the secretion of TNF-α by T cells.

Effect of TS-P and TA-P on RAW264.7 Cells Activity In Vitro
Macrophages are mononuclear phagocytes involved in immune response, inflammation and many homeostasis processes [39]. The analysis of results showed that TS-P and TA-P can significantly promote the proliferation of RAW264.7 cells (p < 0.01) at the final concentrations of 1.25, 2.5, 5 and 10 µg/mL compared with the blank group ( Figure 7A,B). The cell proliferation rate reached 69.30% at the final concentration of 2.5 µg/mL TS-P, which was equivalent to the positive group (65.60%). The maximum proliferation rate was 34.60% when the final concentration of TA-P was 2.5 µg/mL, which was higher than that of the positive group (16.40%). The observation results of cell morphology showed that the RAW264.7 cells in the blank group are round, almost without pseudopodia, and grow on the wall. With the increase in the final concentrations of TS-P and TA-P, the number of cells increased and some cells showed pseudopodia ( Figure 7C,D).
Based on the results of the cell proliferation effects, the optimal concentration 2.5 µg/mL of TS-P and 2.5 µg/mL of TA-P were selected for further study on the effect of TS-P and TA-P on the RAW264.7 cell cycle ( Figure 7E-H). The percentage of RAW264.7 cells in the G0/G1 phase of TS-P and the positive group were significantly reduced by 5.20% and 9.20%, respectively, compared with the blank group. The percentage of RAW264.7 cells in S phase in the TS-P group increased extremely significantly by 3.10% (p < 0.01). The percentage of RAW264.7 cells in the G2/M phase in TS-P and the positive group increased by 1.00% and 14.50% respectively; however, only the G2/M phase of the positive group had statistical significance (p < 0.01). The percentage of RAW264.7 cells in the G0/G1 phase of TA-P and positive group decreased, while the percentage of cells in the S phase and G2/M phase increased. The G0/G1 phase in TA-P group was significantly different from the blank group (p < 0.01) and the number of cells decreased from 52.97% to 50.00%. The percentage of cells of the positive group in the G0/G1 phase decreased by 7.87%, and the percentage of cells in the S phase and G2/M phase increased by 2.84% and 4.80%, respectively. These results show that TS-P and TA-P can affect the macrophage cycle by reducing the preparation time of G0/G1 to promote the proliferation of macrophages.
The secretion amounts of TNF-α under 2.5 µg/mL TS-P, 2.5 µg/mL TA-P and 5 µg/mL LPS were 694.00 pg/mL (p < 0.01), 336.02 pg/mL (p < 0.05) and 14,392.00 pg/mL (p < 0.01), respectively, and the secretion amounts of IL-1β were 31.40 pg/mL, 31.50 pg/mL and 256.00 pg/mL (p < 0.01), respectively. TS-P and TA-P could promote the secretion of TNF-α by macrophages with lower secretion amounts than the positive group but could not promote the secretion of IL-1β ( Figure 7I,J). The percentage of cells of the positive group in the G0/G1 phase decreased by 7.87%, and the percentage of cells in the S phase and G2/M phase increased by 2.84% and 4.80%, respectively. These results show that TS-P and TA-P can affect the macrophage cycle by reducing the preparation time of G0/G1 to promote the proliferation of macrophages. As early as the 1960s, Chihara et al. reported the anti-tumor properties of shiitake mushroom polysaccharides [6]. More and more rare, edible and medicinal fungal polysaccharides in China have been discovered [40,41]. Zhou et al. found that Grifola frondosa polysaccharides could significantly promote macrophage production of NO and secretion of cytokines (TNF)-α, IL-1β and IL-δ [42]. Ding et al. reported that the Gomphus clavatus Gray polysaccharide could induce the apoptosis of HepG-2 cells and affect the mRNA expression of various housekeeping genes in the HepG-2 cells [43]. In our study, the two polysaccharides with novel structures could lead to immune cell proliferation by mainly affecting the G0/G1 phase of the cell cycle, and simultaneously promote the secretion of IgA, IgG and TNF-α by immune cells. Polysaccharide TS-P, which had more branching structures and lower molecular weight, displayed better proliferation effects on three types of cells than TA-P.
The immunoassay showed that TS-P and TA-P could significantly promote the proliferation of B cells, T cells and RAW264.7 cells. The cell proliferation activity of TS-P was better than that of TA-P. The optimal concentration of TS-P to stimulate the proliferation of the three kinds of cells was 2.5 µg/mL, with proliferation rates of 40.50%, 59.30% and 69.00%, respectively. The optimal concentrations of TA-P to stimulate the proliferation of the three kinds of cells are 5, 2.5 and 2.5 µg/mL, with proliferation rates of 29.60%, 43.90% and 34.60%, respectively. The cell cycle results showed that for B cells and macrophages, TS-P and TA-P mainly affected the G0/G1 phase of the cell cycle; for T cells, TS-P affected the G2/M phase and TA-P mainly affected the G0/G1 phase. TS-P can significantly promote B cells to secrete IgA, IgG and IgD (p < 0.01), while TA-P can significantly promote the secretion of IgA and IgG (p < 0.01). The amount of IgD secreted by B cells stimulated by TS-P and the amount of IgA secreted by B cells stimulated by TA-P were both higher than those in the positive group. Both TS-P and TA-P could significantly promote the secretion of TNF-α by T cells (p < 0.01) but only the TA-P group was significantly better than the positive group. Both TS-P and TA-P can promote the secretion of TNF-α by macrophages; the difference between the TS-P group and blank group was very significant (p < 0.01), the difference between the TA-P group and blank group was significant (p < 0.05), and both effects were lower than that of the positive group. Neither of them could promote the secretion of IL-1β by macrophages. The polysaccharide TS-P, which had more branching structures and smaller molecular weight, displayed better proliferation effects on three types of cells than TA-P.
At present, most of the research on edible and medicinal fungi is only at the early stages, but fungi polysaccharides have good biological activity and great application prospects in food and industrial development, which is worthy of further extensive research. Comparative studies on the structure and biological activities of the two new polysaccharides from Tricholoma sinoportentosum (TS-P) and Termitomyces albuminosus (TA-P) could provide certain scientific bases for the in-depth study of polysaccharides.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
All authors declare that there is no conflict of interest.